Polysaccharide degrading enzymes are useful in a variety of applications, such as in animal feed, industrial applications, and, in particular, in ethanol production.
Fossilized hydrocarbon-based energy sources, such as coal, petroleum and natural gas, provide a limited, non-renewable resource pool. Because of the world's increasing population and increasing dependence on energy sources for electricity and heating, transportation fuels, and manufacturing processes, energy consumption is rising at an accelerating rate. The US transportation sector alone consumes over 100 billion gallons of gasoline per year. Most (˜60%) of the oil used in the US today is imported, creating a somewhat precarious situation in today's political climate because supply disruptions are highly likely and would cripple the ability of the economy to function. Fossil petroleum resources, on which our standard of living currently depends, will likely be severely limited within the next 50-100 years.
The production of ethanol from cellulosic biomass can utilize large volumes of agricultural resources that are untapped today. Ethanol is key to partially replacing petroleum resources, which are limited. Ethanol fuels burn cleanly and because of this, ethanol replacement of petroleum fuels at any ratio will have a positive impact on the environment. Production of ethanol from domestic, renewable sources also ensures a continuing supply. For these reasons, the production of ethanol fuels from cellulosic biomass are being developed into a viable industry. High yields of glucose from cellulose (using cellulase enzymes) are required for any economically viable biomass utilization strategy to be realized. The US is one country involved in ethanol production and currently manufactures approximately over three billion gallons of ethanol from corn grain-derived starch. (American Coalition of Ethanol Production, www.ethanol.org; also, Sheehan, J. “The road to bioethanol: A strategic perspective of the US Department of Energy's National Ethanol Program” Himmel M E, Baker J O, Saddler J N eds., Glycosyl Hydrolases for Biomass Conversion, 2-25). Ethanol that is produced from corn starch, however, is limited as an alternative to fossil fuels.
Unharvested residues from agricultural crops are estimated at a mass approximately equal to the harvested portion of the crops. Specifically for the corn crop, if half of the residue could be used as a feedstock for the manufacture of ethanol, then about 120 million tons of corn stover would be available annually for biomass conversion processes (Walsh, Marie E. Biomass Feedstock Availability in the United States. State Level Analysis. 1999). Assuming that mature, dry corn stover is approximately 40% cellulose on a dry weight basis then 48 million tons of cellulose/year would be available for hydrolysis to glucose. Using today's technology, a ton of cellulose will yield approximately 100 gallons of ethanol.
Because known technologies for ethanol production from cellulosic biomass have been more costly than the market price for ethanol, cellulosic ethanol will not become an important alternative to fossil fuels, unless the price of fossil fuels rises substantially. If, however, the cost of the production of ethanol from plant biomass could be reduced, then ethanol might become a cost-effective alternative to fossil fuels even at today's prices for fossil fuels.
Plant biomass is a complex matrix of polymers comprising the polysaccharides cellulose and hemicellulose, and a polyphenolic complex, lignin, as the major structural components. Any strategy designed to substitute cellulosic feedstocks for petroleum in the manufacture of fuels and chemicals must include the ability to efficiently convert the polysaccharide components of plant cell walls to soluble, monomeric sugar streams. Cellulose, the most abundant biopolymer on earth, is a simple, linear polymer of glucose. However, its semi-crystalline structure is notoriously resistant to hydrolysis by both enzymatic and chemical means. Yet, high yields of glucose from cellulose are critical to any economically viable biomass utilization strategy.
Nature has developed effective cellulose hydrolytic machinery, mostly microbial in origin, for recycling carbon from plant biomass in the environment. Without it, the global carbon cycle would not function. To date, many cellulase genes have been cloned and sequenced from a wide variety of bacteria, fungi and plants, and many more certainly await discovery and characterization (Schulein, M, 2000. Protein engineering of cellulases. Biochim. Biophys. Acta 1543:239-252); Tomme P, et al. 1995. Cellulose Hydrolysis by Bacteria and Fungi. Advances in Microbial Physiology 37:1-81). Cellulases are a subset of the glycosyl hydrolase superfamily of enzymes that have been grouped into at least 13 families based on protein sequence similarity, enzyme reaction mechanism, and protein fold motif.
At present enzyme production is primarily by submerged culture fermentation. The scale-up of fermentation systems for the large volumes of enzyme required for biomass conversion would be difficult and extremely capital intensive. For purposes of comparison, a single very large (1 million liter), aerobic fermentation tank could produce 3,091 tons of cellulase protein/yr in continuous culture. Currently, however, fermentation technology is practiced commercially on a significantly smaller scale and in batch mode, so production capacities are closer to 10% of the theoretical 3,091 tons calculated above. Thus, using these assumptions, current practices would yield 3000 times less than the 1.2 MM tons of enzyme needed to convert the cellulose content from 120 MM tons per year of corn stover. Capital and operating costs of such a fermentative approach to producing cellulases are likely to be impractical due to the huge scale and capital investment that will be required.
A system which would reduce the costs associated with providing such enzymes and/or increase production of fermentable sugars produced in saccharification of plant polysaccharides would be highly beneficial.